This invention relates in general to capillary electrophoresis and, in particular, to a capillary electrophoretic system with means for replacing separation medium in a capillary.
High resolution separation of complex mixtures of biomolecules by capillary electrophoresis often requires the use of highly viscous separation media. For example, polyacrylamide gels in the 4-15 percentage T range (either cross-linked or linear) are currently selected to size-separate the products of Sanger-Coulson DNA sequencing reactions. The use of "fixed" polyacrylamide gels which are polymerized within the capillary and are chemically bound (either covalently or by ionic linkage) to the capillary inner walls has been reported. Although impressive resolution has been demonstrated using fixed gels, the use-life of this type of gel is highly variable and is typically quite short.
An alternative to the fixed gel format is described in "Pressure Refilled Polyacrylamide Columns for the Separation of Oligonucleotides by Capillary Electrophoresis," by Sudor, J. et al., Electrophoresis, 1991, 12, 1056-1058. In this approach, the polyacrylamide gel is first polymerized in a syringe and is then driven under high pressure into a wall coated capillary tube. The gel matrix may be replaced under each separation and the column use-life is then determined by the stability of the wall coating rather than by the stability of the fixed gel matrix. The gel is driven by high pressure, such as by hand pressurizing a small volume syringe. Connection of the syringe to the capillary tube was made using either a Teflon sheath or a custom-made tapered glass syringe as described by Sudor et al. The connection was made by hand and had to be made and broken once for each gel replacement. The capillary tube is effectively removed from the electrophoresis circuit during the resealing process.
The replaceable gel currently being produced and used requires 700-1,300 psi pressure to be driven through the capillary tubes with internal diameters of 75-100 microns in a 5-15 minute refill period. This pressure range is far above that which is compatible with easily made and broken seals such as that usually used in capillary electrophoretic systems. Because of this and other factors described above, it is difficult to automate the gel replacement process using conventional or other electrophoretic systems that have been proposed. It is therefore desirable to provide an improved electrophoretic system which avoids the above drawbacks.